This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have developed a pallet array nanotechnology that permits isolation of individual adherent cells. Combining this nanotechnology with antibody-based detection methods, advanced optical imaging and fine needle aspirate (FNA) sampling of breast tumors, we are piloting a methodology that 1) overcomes limitations to existing technologies, such as laser microdissection, 2) could be available to patients at the time of diagnosis vs. after tumor resection, 3) permits enumeration of various cellular elements present within a tumor potentially yielding important information for prognosis or predictive of therapeutic efficacy, 4) is designed for high throughput automated analyses, and 4) has the potential to assess the molecular profile of individual cellular compartments, which could provide additional information for the design of tailored individualized therapy. The HYPOTHESIS for these studies is that the pallet array nanotechnology will permit the identification, enumeration, and isolation of the following individual cellular elements from primary breast tumors: breast cancer stem cells, endothelial progenitor cells, myoepithelial cells, and inflammatory infiltrate. This hypothesis will be tested by pursuing the following Specific Aims: 1. Establish the detection threshold for identifying rare adherent cells. We will employ mixtures of cells expressing unique combinations of cell surface molecules in varying proportions, confirmed by flow cytometry, applied to the pallet array. As detection of multiple tumor cellular subsets will require multicolor/multi-antigen detection, we will perform multi-color fluorescence imaging to establish the detection threshold for rare cells. Additionally, we will refine the design of the pallet array. 2. Apply this methodology to primary breast cancer cells using Fine Needle Aspirate (FNA) sampling. Pilot feasibility studies will be conducted on breast tumor specimens using FNAs performed immediately after resection, in order to test the ability to generalize the findings from cell lines to primary tumor tissues. These studies will establish isolation, identification, and detection methods for the enumeration of breast cancer stem cells, endothelial progenitor cells, myoepithelial cells, tumor epithelial cells, and inflammatory infiltrates.